4-Fluoro-N- indan-2-yl benzamide and its use as a pharmaceutical

ABSTRACT

The present invention relates to the method of stimulating the expression of endothelial NO-synthase in a mammal, which method comprises administering a physiologically active amount of 4-fluoro-N-indan-2-yl benzamide according to the formula (I)  
                 
 
     to the said mammal.  
     The compound (I) can be used for the therapy and prophylaxis of cardiovascular diseases like stable and unstable angina pectoris, Prinzmetal angina (spasm), acute coronary syndrome, heart failure, myocardial infarction, stroke, thrombosis, peripheral artery occlusive disease PAOD, atherosclerosis, restenosis, endothelial damage after PTCA, essential hypertension, pulmonary hypertension, secondary hypertension, renovascular chronic glomerulonephritis, erectile dysfunction, ventricular arrhythmia, and the lowering of cardiovascular risk of postmenopausal women or after intake of contraceptives, the therapy and prophylaxis of diabetes and diabetes complications (nephropathy, retinopathy), angiogenesis, asthma bronchiale, chronic renal failure, cirrhosis of the liver, restricted memory performance or a restricted ability to learn.

[0001] The present invention relates to 4-fluoro-N-indan-2-yl benzamideof the formula (I) and its use as pharmaceutical agent.

[0002] Endothelial NO synthase (eNOS, NOS-III) belongs to a group ofthree isoenzymes which produce nitric oxide (NO) by oxidation ofarginine. Endothelially released NO is of central importance in a numberof key cardiovascular mechanisms. It has a vasodilating effect andinhibits the aggregation of platelets, the adhesion of leukocytes to theendothelium, and the proliferation of intimal smooth muscle cells.

[0003] Endothelial NO synthase is subject to physiological andpathophysiological regulation both at the transcriptional and at thepost-transcriptional level. Enzyme already present in the endotheliummay undergo calcium-dependent and calcium-independent activation throughphosphorylation of specific amino acids, but also by direct interactionswith specific proteins. Stimulators of this, usually transient, NOrelease are extracellular arginine, 17β-estrogen and the mechanicalstimulus exerted on the luminal surface of the endothelium by the bloodflow (shear stress). The latter additionally leads to regulation of eNOSat the transcriptional level. Thus, for example, Sessa et al. (Circ.Research 74 (1994) 349-353) were able by means of exercise training andthe increase in shear stress associated therewith to obtain a markedincrease in eNOS.

[0004] Whether regulation at the post-transcriptional level is relevantin vivo, is not unambiguously proved. Thus, for example, administrationof a high arginine dose is followed by only a transient improvement inthe endothelium-dependent vasorelaxation in patients with coronary heartdisease.

[0005] On the other hand, the significance of the upregulation of theeNOS protein is scientifically accepted. Thus, there are findings whichshow that the protective properties of the HMG-CoA reductase inhibitorsimvastatin can be attributed, besides the lipid lowering, also in partto an increase in eNOS expression in vivo (Endres et al., Proc. Natl.Acad. Sci. USA 95 (1998) 8880-8885). It is additionally known thatsingle point mutations in the 5′-flanking region of the eNOS gene (“eNOSpromoter”), and the reduction in the rate of eNOS gene transcriptionassociated therewith, in the Japanese population is associated with anincrease in the risk of coronary spasms (Nakayama et al., Circulation 99(1999) 2864-2870).

[0006] The current assumption therefore is that the transcriptional andpost-transcriptional mechanisms of eNOS regulation are seriouslydisturbed in a large number of disorders, especially in cardiovasculardisorders. Even in very early stages of a wide variety of cardiovasculardisorders it is possible for a dysfunction of this type in theendothelium lining the blood vessels to lead to a deficiency ofbioactive NO, which is manifested as the disorder progresses in the formof measurable pathophysiological and morphological changes. Thus,critical steps in early atherogenesis are speeded up by a decrease inendothelial NO release, such as, for example, the oxidation of lowdensity lipoproteins, the recruitment and deposition of monocytes in theintima of vessels, and the proliferation of intimal cells. A consequenceof atherogenesis is the formation of plaques on the inside of the bloodvessels, which may in turn lead, through a diminution in the shearstress, to a further decrease in endothelial NO release and a furtherdeterioration in the pathology. Since endothelial NO is also avasodilator, a decrease thereof frequently also leads to hypertension,which may, as an independent risk factor, cause further organ damage.

[0007] The aim of a therapeutic approach to the treatment of thesedisorders must accordingly be to interrupt this chain of events byincreasing the endothelial NO expression. Gene transfer experimentswhich lead in vitro to overexpression of NO synthase in previouslydamaged vessels are in fact able to counteract the described processesand are thus evidence of the correctness of this approach (Varenne etal., Hum. Gene Ther. 11 (2000) 1329).

[0008] Some low molecular weight compounds which, in cell cultures, maylead to a direct effect on eNOS transcription and expression aredisclosed in the literature. The statins which have already beenmentioned are, however, the only substances for which it has beenpossible to date to show such an increase in eNOS in vivo as a sideeffect. In view of the known range of side effects of this class ofsubstances, however, it is unclear how far this effect is present in atoxicologically unproblematic dose.

[0009] Liao et al. claim in WO 99/47153 and WO 00/03746 the use ofrhoGTPase inhibitors and agents which influence the organization of theactin cytoskeleton for increasing eNOS in endothelial cells and for thetherapy of various disorders such as, for example, strokes or pulmonaryhypertension, without, however, indicating a specific way of achievingthis.

[0010] Thus, there exists a strong need for a medicament whichupregulates eNOS-expression in endothelial cells. The object of thepresent invention is to provide a compound showing this ability.

[0011] This object is attained by the use of 4-fluoro-N-indan-2-ylbenzamide according to the formula (I)

[0012] for the manufacture of a medicament for the stimulation of theexpression of endothelial NO synthase.

[0013] This object is also attained by a pharmaceutical preparation forthe stimulation of the expression of endothelial NO synthase comprisingan effective dose of 4-fluoro-N-indan-2-yl benzamide and apharmaceutically acceptable carrier.

[0014] The present invention furthermore includes the use of allsolvates of the compound according to formula (I), for example,hydrates, adducts with alcohols, active metabolites of the compound offormula (I), and also derivatives and prodrugs of the compound offormula (I) which contain physiologically tolerable and cleavablegroups, for example, esters, amides and compounds in which the N-H groupdepicted in formula (I) is replaced with a N-alkyl group, such asN-methyl, or with a N-acyl group, such as N-acetyl or N-argininyl,including pharmaceutically acceptable salts formed on functional groupspresent in the N-acyl group.

[0015] WO 00/51970 discloses the manufacture and use of4-fluoro-N-indan-2-yl benzamide as a medicament. The compound possessesstrong potentiation of the cholinergic activity, and is useful for thetreatment and/or prevention of disorders in the central nervous systemfor mammals, and more particularly of amnesia, dementia, e.g. seniledementia, Alzheimer's dementia, dementia associated with variousdiseases such as cerebral vascular dementia, cerebral post-traumaticdementia, dementia due to brain tumor, dementia due to chronic subduralhematoma, dementia due to normal pressure hydrocephalus, post-meningitisdementia, Parkinson's disease type dementia, and the like. The compoundis expected to be useful as a therapeutic and/or preventive agent forschizophrenia, depression, stroke, head injury, nicotine withdrawal,spinal cord injury, anxiety, pollakiuria, incontinence of urine,myotonic dystrophy, attention deficit hyperactivity disorder, excessivedaytime sleepiness (narcolepsy), Parkinson's disease or autism. WO00/51970 does not disclose or suggest the use of 4-fluoro-N-indan-2-ylbenzamide for the upregulation of the expression of endothelicalNO-synthase, in particular for the treatment of cardiovascular diseases,stable or unstable angina pectoris, coronary heart disease, Prinzmetalangina, acute coronary syndrome, heart failure, myocardial infarction,stroke, thrombosis, peripheral artery occlusive disease, endothelialdysfunction, atherosclerosis, restenosis, endothelial damage after PTCA,hypertension, essential hypertension, pulmonary hypertension, secondaryhypertension, renovascular hypertension, chronic glomerulonephritis,erectile dysfunction, ventricular arrhythmia, diabetes or diabetescomplications, nephropathy, retinopathy, angiogenesis, asthmabronchiale, chronic renal failure, cirrhosis of the liver, osteoporosis,restricted memory performance, a restricted ability to learn, or thelowering of cardiovascular risk of postmenopausal women or after intakeof contraceptives.

[0016] The compound according to formula (I) can be prepared startingfrom 2-indanylamine which is known in the literature. 2-Indanyl aminecan be reacted, in the form of the free base or a salt thereof, with4-fluorobenzoylchloride in the presence of a base like, for example,triethylamine. The reaction is generally carried out in a solvent likedichloromethane, tetrahydrofuran, toluene or dioxane, and preferably atroom temperature. Alternatively, the compound according to the formula(I) is obtained by a coupling reaction of the said 2-indanyl amine with4-fluorobenzoic acid, in the presence of a base like, for example,diisopropylethylamine, and the use of an appropriate coupling reagentlike, for example, carbodiimides, HATU or TOTU.

[0017] Further reactions for the synthesis of the compound according tothe formula (I) are apparent to or well-known to the skilled person andcan be carried out under standard conditions according to or analogouslyto procedures described in the literature, for example in Houben-Weyl,Methoden der Organischen Chemie (Methods of Organic Chemistry),Thieme-Veriag, Stuttgart, or Organic Reactions, John Wiley & Sons, NewYork. If desired, the compound of the formula (I) can be purified bycustomary purification procedures, for example by recrystallization orchromatography. The starting compounds for the preparation of thecompound of the formula (I) are commercially available or can beprepared according to or analogously to literature procedures.

[0018] The compound according to the formula (I) can be used toupregulate the expression of the endothelial NO synthase and is a usefulpharmaceutical compound for the treatment of various diseases. In thecontext of the present invention, treatment includes the therapy as wellas the prophylaxis of the respective diseases.

[0019] Examples of diseases which can be treated with the compound (I)according to the present invention include cardiovascular diseases likestable and unstable angina pectoris, coronary heart disease, Prinzmetalangina (spasm), acute coronary syndrome, heart failure, myocardialinfarction, stroke, thrombosis, peripheral artery occlusive disease(PAOD), endothelial dysfunction, atherosclerosis, restenosis,endothelial damage after PTCA, hypertension including essentialhypertension, pulmonary hypertension, and secondary hypertension(renovascular hypertension, chronic glomerulonephritis), erectiledysfunction, ventricular arrhythmia, and the lowering of cardiovascularrisk of postmenopausal women or after intake of contraceptives.

[0020] Compound (I) can additionally be used in the therapy andprophylaxis of diabetes and diabetes complications (nephropathy,retinopathy), angiogenesis, asthma bronchiale, chronic renal failure,cirrhosis of the liver, osteoporosis, restricted memory performance or arestricted ability to learn.

[0021] Preferred indications are stable angina pectoris, coronary heartdisease, hypertension, endothelial dysfunction, atherosclerosis anddiabetes complications.

[0022] The compound according to the formula (I) can also be used incombination with other pharmaceutically active compounds, preferablycompounds which are able to enhance the effect of the compound accordingto the formula (I). Examples of such compounds include: statins;ACE-inhibitors; AT1-antagonists; argininase-inhibitors; PDEV-inhibitors; Ca-antagonists; alpha-blockers; beta-blockers; metimazoland analogous compounds; arginine; tetrahydrobiopterin; vitamins, inparticular vitamin C and vitamin B6; niacin.

[0023] The compound (I), optionally in combination with otherpharmaceutically active compounds, can be administered to animals,preferably to mammals, and in particular to humans, as a pharmaceuticalby itself, or in the form of pharmaceutical preparations. Furtherobjects of the present invention are pharmaceutical preparations (orpharmaceutical compositions) which comprise an effective dose of thecompound of the formula (I) and a pharmaceutically acceptable carrier,i.e., one or more pharmaceutically acceptable carrier substances and/oradditives. Said pharmaceutical preparations are used for stimulating theexpression of endothelial NO synthase and in particular a medicament forthe therapy and prophylaxis of the above-mentioned syndromes.

[0024] The pharmaceutical according to the invention can be administeredorally, for example, in the form of pills, tablets, lacquered tablets,sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous,alcoholic or oily solutions, syrups, emulsions or suspensions, orrectally, for example, in the form of suppositories. Administration canalso be carried out parenterally, for example, subcutaneously,intramuscularly or intravenously in the form of solutions for injectionor infusion. Other suitable administration forms are, for example,percutaneous or topical administration, for example, in the form ofointments, tinctures, sprays or transdermal therapeutic systems, or theinhalative administration in the form of nasal sprays or aerosolmixtures, or, for example, microcapsules, implants or rods. Thepreferred administration form depends, for example, on the disease to betreated and on its severity.

[0025] The amount of the compound of the formula (I) in thepharmaceutical preparations normally ranges from 0.2 to 800 mg,preferably from 0.5 to 500 mg, in particular from 1 to 200 mg, per dose,but depending on the type of the pharmaceutical preparation it may alsobe higher. The pharmaceutical preparations usually comprise 0.5 to 90percent by weight of the compound of the formula (I). The preparation ofthe pharmaceutical preparations can be carried out in a manner known perse. To this end, the compound of the formula (I), together with one ormore solid or liquid pharmaceutical carrier substances and/or additives(or auxiliary substances) and, if desired, in combination with otherpharmaceutically active compounds having therapeutic or prophylacticaction, are brought into a suitable administration form or dosage formwhich can then be used as a pharmaceutical in human or veterinarymedicine.

[0026] For the production of pills, tablets, sugar-coated tablets andhard gelatin capsules it is possible to use, for example, lactose,starch, for example, maize starch, or starch derivatives, talc, stearicacid or its salts, etc. Carriers for soft gelatin capsules andsuppositories are, for example, fats, waxes, semisolid and liquidpolyols, natural or hardened oils, etc. Suitable carriers for thepreparation of solutions, for example of solutions for injection, or ofemulsions or syrups are, for example, water, physiological sodiumchloride solution, alcohols such as ethanol, glycerol, polyols, sucrose,invert sugar, glucose, mannitol, vegetable oils, etc. It is alsopossible to lyophilize the compound of the formula (I) and to use theresulting lyophilizates, for example, for preparing preparations forinjection or infusion. Suitable carriers for microcapsules, implants orrods are, for example, copolymers of glycolic acid and lactic acid.

[0027] Besides the compound (I) and carriers, the pharmaceuticalpreparations can also contain additives, for example, fillers,disintegrants, binders, lubricants, wetting agents, stabilizers,emulsifiers, dispersants, preservatives, sweeteners, colorants,flavorings, aromatizers, thickeners, diluents, buffer substances,solvents, solubilizers, agents for achieving a depot effect, salts foraltering the osmotic pressure, coating agents or antioxidants.

[0028] The dosage of the compound of the formula (I) to be administereddepends on the individual case and is, as is customary, to be adapted tothe individual circumstances to achieve an optimum effect. Thus, itdepends on the nature and the severity of the disorder to be treated,and also on the sex, age, weight and individual responsiveness of thehuman or animal to be treated, on the duration of action of the compound(I), on whether the therapy is acute or chronic or prophylactic, or onwhether other active compounds are administered in addition to thecompound of the formula (I). In general, a daily dose of approximately0.01 to 100 mg/kg, preferably 0.1 to 10 mg/kg, in particular 0.3 to 5mg/kg (in each case mg per kg of bodyweight) is appropriate foradministration to an adult weighing approximately 75 kg in order toobtain the desired results. The daily dose can be administered in asingle dose or, in particular when larger amounts are administered, bedivided into several, for example two, three or four individual doses.In some cases, depending on the individual response, it may be necessaryto deviate upwards or downwards from the given daily dose.

[0029] The compound according to the formula (I) can also be used forpurposes other than those indicated in the foregoing. Non-limitingexamples include diagnostic purposes, the use as biochemical tools, andas intermediates for the preparation of further compounds, e.g.pharmaceutically active compounds.

[0030] The present invention will now be illustrated in the followingexample:

EXAMPLE Preparation of 4-Fluoro-N-(indan-2-yl)-benzamide

[0031] 43.70 g (258 mol) 2-aminoindane hydrochloride and 53.43 g (528mmol) triethylamine were mixed with 250 ml of tetrahydrofuran, 42.89 g(270 mmol) 4-fluorobenzoylchloride were added, and the mixture wasstirred for 2 h at RT.

[0032] The resulting mixture was then poured onto an ice/HCl-mixture,the obtained precipitate was filtered, washed with a NaHCO₃-solution andwater and dried in vacuo. The crude product was crystallized frommethanol. There were obtained 47.8 g (73%) of a white, crystallineproduct.

[0033] mp.:167° C.

[0034] MS: M+H⁺:256.1

[0035]¹H-NMR (300 MHz, d₆-DMSO): 2.96 (dd, 2H, H1/H3), 3.25 (dd, 2H,H3/H1), 4.70 (sextett, 1H, H2), 7.12-7.19 (m, 2H, H4,7/5,6), 7.20-7.28(m, 2H, H5,6/4,7), 7.30 (t,2H, H3′,5′), 7.95 (dd, 2H, H2′,6′), 8.68 (d,1H, NH)

[0036] Measurement of Activation of eNOS Transcription

[0037] Activation of eNOS transcription was measured as described indetail in Li et al. “Activation of protein kinase C alpha and/or epsilonenhances transcription of the human endothelial nitric oxide synthasegene”, Mol. Pharmacol. 1998, 53: 630-637.

[0038] Briefly, a 3.5 kB long fragment 5′ of the starting codon of theeNOS gene was cloned, sequenced and cloned in firefly luciferaseexpression plasmids to monitor activation of the eNOS promoter byreporter gene activity. A human endothelial cell line stable transfectedand expressing this promoter-reporter construct was used for compoundtesting. Cells were incubated for 18 h with the compound (I).

[0039] Prior to incubation of the cells, the compound (I) was dissolvedin sterile DMSO. A final concentration of 0.5% DMSO in complete mediumwas obtained. Induction of reporter gene expression in these cells wasmeasured using a standard luciferase assay system (Promega, Cat. NoE150) according to the manufacturer's instructions. Luciferase inductionin cells incubated with compound (I) were compared to those incubatedwith solvent alone. The ratio of both activities (transcriptioninduction ratio, TIR) was plotted as a function of compoundconcentration. Typically, TIR values started at low concentrations at aratio of 1, indicating no compound effect, and extended up to a maximumTIR value TIR(max) which indicates the increase of the eNOStranscription. EC₅₀ values of transcription induction ratios as afunction of compound concentration were determined graphically.

[0040] The effect of the compound (I) on eNOS-transcription wasconfirmed in a second assay based on eNOS protein detection. Primaryhuman umbilical vein cord endothelial cells (HUVEC) were isolated andcultivated according to standard procedures. Confluent cells wereincubated with compound (I) for 18 h and the effect on eNOS proteinexpression determined by a quantitative Western blotting procedure.After incubation of compound (I), HUVEC were lysed in ice-cold lysisbuffer containing 10 mM Tris-HCl, pH 8.0, 1% SDS and proteaseinhibitors. The lysate was subjected to a standard denaturatingpolyacrylamid gel electropheresis and blotted to nitrocellulosemembranes. Using a specific primary monoclonal antibody (TransductionLaboratories, UK) and alkaline phosphatase labelled secondary antibody(Jackson Labs), a specific eNOS protein band was visualized andquantified based on a chemifluorescence detection method.

[0041] For the compound (I), the EC₅₀-value was 0.8 μM, theTIR(max)-value was 4.10.

[0042] Animal Models

[0043] All animal experiments were performed in accordance to the Germananimal protection law and to the guidelines for the use of experimentalanimals as given by the Guide for the Care and Use of Laboratory Animalsof the U.S. National Institutes of Health.

[0044] Animals and Treatment (Experiments A-C)

[0045] ApoE and eNOS deficient mice (C57BL/6J background, JacksonLaboratory, Bar Harbor, Me.) were used. All animals were 10-12 weeks ofage and weighed 22 to 28 g. Three days before surgery, mice were dividedinto 4 groups (apoE control, n=10-12; apoE with compound (I), n=10-12;eNOS control, n=10-12; eNOS with compound (I), n=10-12) and receivedeither a standard rodent chow (containing 4% fat and 0,001% cholesterol;in the following designated as placebo group) or a standard rodentchow+compound (I) (10 or 30 mg/kg/d p.o.).

[0046] A Anti-hypertensive Effect in ApoE Knockout Mice

[0047] Blood-pressure was determined in conscious mice using acomputerized tail-cuff system.

[0048] For compound (I), after 4 months treatment of ApoE deficient miceblood pressure was significantly (p<0.05) lowered in the 30 mg/kg/dgroup compared to placebo treatment (92±5 mmHg versus 115±2 mmHg). Noblood pressure reduction could be observed at similar dosing in eNOSdeficient mice after 4 weeks treatment.

[0049] B Inhibition of Neointima Formation and Atherogenesis (FemoralArtery Cuff)

[0050] After 3 days treatment of ApoE deficient mice with compound (I),(10 mg/kg/d pressed in chow), animals were anesthetized with anintraperitoneal injection of pentobarbital (60 mg/kg) followed by anintramuscular injection of xylazin (2 mg/kg) and a cuff was placedaround the femoral artery as described in Moroi et at. (J Clin Invest.101:1225-32, 1998). Briefly, the left femoral artery was dissected. Anon-occlusive 2.0 mm polyethylene cuff made of PE-50 tubing (innerdiameter 0.56 mm, outer diameter 0.965 mm, Becton Dickinson, MountainView, Calif.) was placed around the artery and tied in place with two7-0 sutures. The right femoral artery was isolated from the surroundingtissues but a cuff was not placed. Treatment with compound (I) wascontinued for 14 days after surgery. Then the animals were sacrificed.The aortas were taken for determination of vascular eNOS expressions byquantitative western blotting. Both femoral arteries were harvested,fixed in formalin and embedded in paraffin. 20 cross sections (10 μm)were cut from the cuffed portion of the left femoral artery and from thecorresponding segment of the right artery. Sections were subjected tostandard hematoxylin and eosin staining. Morphometric analyses wereperformed using an image analysis computer program (LeicaQWin, LeicaImaging Systems, Cambridge, GB). For each cross section the area of thelumen, the neointima and the media were determined. To this end, theneointima was defined as the area between the lumen and the internalelastic lamina and the media was defined as the area between theinternal and the external elastic lamina. The ratio between the area ofthe neointima and the area of the media was expressed as theneointima/media ratio.

[0051] Compound (I) reduced the maladaptive neo-intima formation by afactor of 2, decreasing the neointima to media ratio from 0.39±0.07 inthe placebo group to 0.170±0.04 in the compound group. In parallel,vascular eNOS expression was enhanced by a factor of 2.1. No effect ofcompound (I) could be demonstrated in a similar setup using eNOSdeficient mice instead of ApoE knockout mice.

[0052] C Prevention of Atherosclerotic Plaque Formation in ChronicTreatment

[0053] ApoE deficient mice were treated for 16 weeks with compound (I)pressed in chow and finally sacrificed. Aortas were removed from eachmouse, fixed in formalin and embedded in paraffin. Plaque formation wasmeasured via lipid lesions formation in the aortas (from aortic arch todiaphragm) and was analyzed by oil red O staining. For quantifying theeffect of the respective compound on vascular eNOS expression thefemoral arteries were used in this experiment.

[0054] Compound (I) according to the present invention significantlyreduced plaque formation (5.2±1% versus 13.3±2.6 in the placebo group,values in overall plaque size in % of total surface). Vascular eNOSexpression was found to be 1.75 fold up-regulated in the treatmentgroup.

[0055] D Improvement of Coronary Function in Diseased ApoE DeficientMice

[0056] Old Male wild-type C57BL/6J mice (Charles River Wiga GmbH,Sulzfeld), and apo1E deficient mice (C57BL/6J background, JacksonLaboratory, Bar Harbor, Me.) 6 month of age and weighing 28 to 36 g wereused in the experiments. Mice were divided into 3 groups (C57BL/6, n=8;apoE control, n=8; apoE with compound (I), n=8) and received for 8 weekseither a standard rodent chow (containing 4 % fat and 0,001%cholesterol) or a standard rodent chow+compound (I) (30 mg/kg/d p.o.).

[0057] Mice were anesthetized with sodium pentobarbitone (100 mg/kgi.p.), and the hearts were rapidly excised and placed into ice-coldperfusion buffer. The aorta was cannulated and connected to a perfusionapparatus (HUGO SACHS ELECTRONICS, Freiburg, Germany) which was startedimmediately at a constant perfusion pressure of 60 mm Hg. Hearts wereperfused in a retrograde fashion with modified Krebs bicarbonate buffer,equilibrated with 95% O₂ and 5% CO₂ and maintained at 37.5° C.

[0058] A beveled small tube (PE 50) was passed through a pulmonary veininto the left ventricle and pulled through the ventricular wall,anchored in the apex by a fluted end, and connected to atip-micromanometer (Millar 1.4 French). The left atrium was cannulatedthrough the same pulmonary vein and the heart switched to the workingmode with a constant preload pressure of 10 mm Hg and an afterloadpressure of 60 mm Hg. Aortic outflow and atrial inflow were continuouslymeasured using ultrasonic flow probes (HSE/Transonic Systems Inc.).Coronary flow was calculated as the difference between atrial flow andaortic flow. All hemodynamic data were digitized at a sampling rate of1000 Hz and recorded with a PC using specialized software (HEM,Notocord).

[0059] Hearts were allowed to stabilize for 30 min. All functionalhemodynamic data were measured during steady state, and during volume-and pressure loading.

[0060] Left ventricular function curves were constructed by varyingpre-load pressure. For acquisition of preload curves, afterload was setat 60 mm Hg and preload was adjusted in 5 mm Hg steps over a range of 5to 25 mm Hg. Hearts were allowed to stabilize at baseline conditionsbetween pressure- and volume-loading.

[0061] Isolated hearts from ApoE deficient animals displayed a lowercoronary flow in this setup compared to C57B16 wildtype mice (3.6 ml/minversus 4.95 ml/min). Treatment of ApoE deficient animals with thecompound (I) according to the present invention increased coronary flowto 5 ml/min comparable to the levels of non-diseased wildtype mice.Compound (I) also improved pre-load dependent coronary flow and reducedthe incidence of ventricular arrhythmias as an indicator foranti-ischemic efficacy.

We claim:
 1. A method of stimulating the expression of endothelialNO-synthase in a mammal, which method comprises administering aphysiologically active amount of 4-fluoro-N-indan-2-yl benzamideaccording to the formula (I)

to the said mammal.
 2. The method of claim 1, wherein the mammal is ahuman.
 3. A method of treating a disease from the group consisting ofcardiovascular diseases, stable and unstable angina pectoris, coronaryheart disease, Prinzmetal angina, acute coronary syndrome, heartfailure, myocardial infarction, stroke, thrombosis, peripheral arteryocclusive disease, endothelial dysfunction, atherosclerosis, restenosis,endothelial damage after PTCA, hypertension, essential hypertension,pulmonary hypertension, secondary hypertension, renovascularhypertension, chronic glomerulonephritis, erectile dysfunction,ventricular arrhythmia, diabetes and diabetes complications, nephropathyand retinopathy, angiogenesis, asthma bronchiale, chronic renal failure,cirrhosis of the liver, osteoporosis, restricted memory performance, arestricted ability to learn, and the lowering of cardiovascular risk ofpostmenopausal women and after intake of contraceptives in a mammal,which method comprises administering a physiologically active amount4-fluoro-N-indan-2-yl benzamide according to the formula (I)

to the said mammal.
 4. The method of claim 1, wherein the mammal is ahuman.
 5. The method according to claim 4, wherein a disease from thegroup consisting of endothelial dysfunction, hypertension, coronaryheart disease, stable angina pectoris, diabetes complications andatherosclerosis is treated.
 6. A pharmaceutical preparation for thestimulation of the expression of endothelial NO-synthase whichpreparation comprises an effective dose of 4-fluoro-N-indan-2-ylbenzamide and a pharmaceutically acceptable carrier.
 7. A pharmaceuticalpreparation according to claim 6, which pharmaceutical preparation is inthe form of a pill, tablet, lacquered tablet, sugar-coated tablet,granule, hard or soft gelatin capsule, aqueous, alcoholic or oilysolution, syrup, emulsion or suspension, suppository, solution forinjection or infusion, ointment, tincture, spray, transdermaltherapeutic system, nasal spray, aerosol mixture, microcapsule, implantor rod.
 8. A pharmaceutical preparation for the treatment of a diseasefrom the group consisting of cardiovascular diseases, stable andunstable angina pectoris, coronary heart disease, Prinzmetal angina,acute coronary syndrome, heart failure, myocardial infarction, stroke,thrombosis, peripheral artery occlusive disease, endothelialdysfunction, atherosclerosis, restenosis, endothelial damage after PTCA,hypertension, essential hypertension, pulmonary hypertension, secondaryhypertension, renovascular hypertension, chronic glomerulonephritis,erectile dysfunction, ventricular arrhythmia, diabetes and diabetescomplications, nephropathy and retinopathy, angiogenesis, asthmabronchiale, chronic renal failure, cirrhosis of the liver, osteoporosis,restricted memory performance, a restricted ability to learn, and thelowering of cardiovascular risk of postmenopausal women and after intakeof contraceptives, which preparation comprises an effective dose of4-fluoro-N-indan-2-yl benzamide and a pharmaceutically acceptablecarrier.
 9. A pharmaceutical preparation according to claim 8, whereinthe disease to be treated is from the group consisting of endothelialdysfunction, hypertension, coronary heart disease, stable anginapectoris, diabetes complications and atherosclerosis.
 10. Apharmaceutical preparation according to claim 8, which pharmaceuticalpreparation is in the form of a pill, tablet, lacquered tablet,sugar-coated tablet, granule, hard or soft gelatin capsule, aqueous,alcoholic or oily solution, syrup, emulsion or suspension, suppository,solution for injection or infusion, ointment, tincture, spray,transdermal therapeutic system, nasal spray, aerosol mixture,microcapsule, implant or rod.